J. Clifford Murray

Nanomedicine: current status and future prospects

Applications of nanotechnology for treatment, diagnosis, monitoring, and control of biological systems has recently been referred to as “nanomedicine” by the National Institutes of Health. Research into the rational delivery and targeting of pharmaceutical, therapeutic, and diagnostic agents is at the forefront of projects in nanomedicine. These involve the identification of precise targets (cells and receptors) related to specific clinical conditions and choice of the appropriate nanocarriers to achieve the required responses while minimizing the side effects. Mononuclear phagocytes, dendritic cells, endothelial cells, and cancers (tumor cells, as well as tumor neovasculature) are key targets. Today, nanotechnology and nanoscience approaches to particle design and formulation are beginning to expand the market for many drugs and are forming the basis for a highly profitable niche within the industry, but some predicted benefits are hyped. This article will highlight rational approaches in design and surface engineering of nanoscale vehicles and entities for site‐specific drug delivery and medical imaging after parenteral administration. Potential pitfalls or side effects associated with nanoparticles are also discussed.—Moghimi, S. M. Hunter, A. C., Murray, J. C. Nanomedicine: current status and future prospects. FASEB J. 19, 311‐330 (2005)

Low and high molecular weight poly(L-lysine)s/poly(L-lysine)-DNA complexes initiate mitochondrial-mediated apoptosis differently.

Poly(l‐lysine)s, PLLs, are commonly used for DNA compaction and cell transfection. We report that, although PLLs of low (2.9 kDa), L‐PLL, and high (27.4 kDa), H‐PLL, Mw in free form and DNA‐complexed cannot only cause rapid plasma membrane damage in human cell lines, phosphatidylserine “scrambling” and loss of membrane integrity, but later (24 h) initiate stress‐induced cell death via mitochondrial permeabilization without the involvement of processed caspase‐2. Mitochondrially mediated apoptosis was confirmed by detection of cytochrome c (Cyt c) release, activation of caspases‐9 and ‐3, and subsequent changes in mitochondrial membrane potential. Plasma membrane damage and apoptosis were most prominent with H‐PLL. Cytoplasmic level of Cyt c was more elevated following H‐PLL treatment, but unlike L‐PLL case, inhibition of Bax channel‐forming activity reduced the extent of Cyt c release from mitochondria by half. Inhibition of Bax channel‐forming activity had no modulatory effect on L‐PLL‐mediated Cyt c release. Further, functional studies of isolated mitochondria indicate that H‐PLL, but not L‐PLL, can directly induce Cyt c release, membrane depolarization, and a progressive decline in the rate of uncoupled respiration. Combined, our data suggest that H‐PLL and L‐PLL are capable of initiating mitochondrially mediated apoptosis differently. The observed PLL‐mediated late‐phase apoptosis may provide an explanation for previously reported transient gene expression associated with PLL‐based transfection vectors. The importance of our data in relation to design of novel and safer cationic non‐viral vectors for human gene therapy is discussed.

Immunohistochemical analysis of endothelial-monocyte-activating polypeptide-II expression in vivo.

Endothelial-monocyte activating polypeptide (EMAP)-II is a novel molecule with cytokine-like pro-inflammatory properties, inducing procoagulant activity on the surface of endothelial cells and monocyte/macrophages in vitro, as well as up-regulating E- and P-selectin expression. EMAP-II is chemotactic for monocytes/macrophages and neutrophils, and stimulates myeloperoxidase release from neutrophils. Injection of EMAP-II into the mouse footpad induces an acute inflammatory response, although some regression occurs in response to direct injection of EMAP-II into murine tumors. Very little is known about the expression of EMAP-II in normal tissues of mice or humans, or about its function in vivo. We developed polyclonal antibodies against EMAP-II using recombinant protein produced in Escherichia coli, and used these antibodies to carry out an immunohistochemical study of the occurrence and distribution of EMAP-II in human tissues. The distribution of EMAP-II protein is relatively restricted, occurring primarily in endocrine organs, in cells of neuroendocrine origin, but also in tissues with high turnover. EMAP-II is strongly expressed in secretory epithelial cells of the thyroid, pancreas, adrenal and salivary glands, among others, as well as in neurons and subsets of monocytes/macrophages. It is also found in the epithelium of the small and large intestines. We conclude that EMAP-II expression is usually, but not always, associated with tissues that display high turnover and high levels of protein synthesis.

Colorectal Cancer Cells Induce Lymphocyte Apoptosis by an Endothelial Monocyte-Activating Polypeptide-II-Dependent Mechanism

Endothelial monocyte-activating polypeptide-II (EMAP-II) was first isolated from cell growth medium conditioned by tumor cells, and is closely related or identical with the p43 component of the mammalian multisynthase complex. In its secreted form, EMAP-II has multiple cytokine-like activities in vitro, inducing procoagulant activity on the surface of endothelial cells, increasing expression of E- and P-selectins and TNF-R1, and directing migration of monocytes and neutrophils. EMAP-II has also been shown to induce apoptosis in endothelial cells, leading to the suggestion that it is a proinflammatory polypeptide with antiangiogenic activity. The role of secreted EMAP-II in tumors remains poorly understood, and we hypothesized that EMAP-II may play a role in immune evasion by tumor cells. We investigated its effects on lymphocytes, using recombinant protein, or colorectal cancer cell lines, as a source of native EMAP-II. Recombinant EMAP-II inhibits DNA synthesis and cell division, and induces apoptosis in mitogen-activated lymphocytes in PBMC preparations, and in Jurkat T cells. Native EMAP-II, released by or expressed on the surface of colorectal carcinoma cells, also induces activation of caspase 8 and apoptosis of PBLs and Jurkat cells, which are partially blocked by addition of Abs against EMAP-II. Thus, activated lymphocytes, along with proliferating endothelial cells, are targets for the cytotoxic activity of EMAP-II. Membrane-bound and soluble EMAP-II appear to play multiple roles in the tumor microenvironment, one of which is to assist in immune evasion.

Endothelial CD44H mediates adhesion of a melanoma cell line to quiescent human endothelial cells in vitro

A critical step in the metastatic spread of tumour cells is the interaction of circulating tumour cells with the vascular endothelium. We have investigated the role of CD44 and its variants in the adhesion of a human melanoma cell line (RPMI‐7951) and a breast adenocarcinoma cell line (MDA‐MB‐231) to quiescent human umbilical vein endothelial cells (HUVEC) in vitro. Both tumour cell lines express CD44H, CD44A and CD44v9, while HUVEC express only CD44H. Pre‐treatment of endothelial cell monolayers with a blocking monoclonal antibody against CD44H (MAb 5A4) reduced the adhesion of RPMI‐7951 cells but not that of MDA‐MB‐231. In contrast, pre‐treatment of both tumour cell lines with the same antibody had no effect on adhesion. Digestion of the CD44 ligand hyaluronic acid (HA) on RPMI‐7951 cells significantly reduced adhesion to endothelial monolayers, while digestion of HUVEC HA had no effect. We conclude that CD44H expressed on the surface of quiescent endothelial monolayers mediates in part the adhesion of the metastatic melanoma cell line RPMI‐7951 but not that of a breast adenocarcinoma line. It does so by acting as a receptor for HA on the tumour cell surface. Tumour cell CD44H and variants CD44A and CD44v9 do not appear to be involved in adhesion to endothelial cells.

Modulation of human endothelial cell procoagulant activity in tumour models in vitro

Several tumour‐derived factors have recently been identified which induce tissue factor (TF) expression in endothelial cells in vitro. However, there is only limited evidence that endothelial cells lining tumour blood vessels express elevated procoagulant activity (PCA) in vivo. We have investigated the effects of human breast and small cell lung cancer cell lines on the PCA of human micro‐ and macrovessel endothelial cell monolayers using a one‐stage clotting assay, as well as detection of TF mRNA by RT‐PCR. Only conditioned medium from the MDA‐MB‐231 breast adenocarcinoma cell line produced a consistent although transient increase in endothelial cell surface PCA, which was maximal by 6–9 hr. TF mRNA was detectable in the endothelial cells after 1 hr incubation with MDA‐MB‐231‐conditioned medium and subsequently fell below detectable levels. Following 24 hr stimulation, nearly half the endothelial cell PCA was due to the presence of TF‐containing membrane vesicles shed by the MDA‐MB‐231 cells. Consistent with these findings, the MDA‐MB‐231 cell line expressed high levels of cell surface‐associated TF activity. Co‐culture of MDA‐MB‐231 and endothelial cells for up to 5 days increased (approx. 118‐fold) PCA associated with endothelial cell monolayers, due mainly to sequestration of shed tumour cell vesicles. Our results suggest that induction of TF de novo is not a common feature in the supporting endothelium of these tumour types.

A simple fluorometric assay for quantifying the adhesion of tumour cells to endothelial monolayers

A static adhesion assay employing 6-carboxy-3′,6′-diacetylfluorescein (6-CFDA) as a fluorescent marker has been developed to study the interactions of tumour cell lines with endothelial monolayers. This assay allows simple, safe quantification of cell-cell adhesion using living cells. It has been used to demonstrate that the integrin adhesion molecule VLA-4 mediates the attachment of RPMI-7951 melanoma cells to human umbilical vein endothelial cells (HUVEC) which have been activated by TNFα. In addition, MDA-MB-231 breast adenocarcinoma cells display greater adhesion to microvessel endothelial cells than to large vessel endothelial cells.

Endothelial monocyte-activating polypeptide-2 is increased in pregnancy but is not further increased in preeclampsia.

Objectives: Endothelial monocyte-activating polypeptide-2 (EMAP-2) is a novel protein that demonstrates potent proinflammatory activity in vivo and in vitro. The purpose of this study was to investigate the expression of EMAP-2 in normal pregnancy and in pregnancies complicated with preeclampsin and to test the hypothesis that EMAP-2 is a causative agent of the endothelial activation of preeclampsia. Methods: Expression of EMAP-2 in the placenta was investigated using reverse transcriptase-polymerase chain reaction to detect mRNA, and immunohistochemistry with an EMAP-2-specific polyclonal antiserum was carried out to detect protein. Levels of circulating EMAP-2 protein were measured in the blood of nonpregnant, normal pregnant, and preeclamptic individuals using a specific enzyme-linked immunoabsorbent assay and the molecular forms present assessed by Western blotting. Results: EMAP-2 is transcribed and translated by the placenta thoughout pregnancy and in preeclampsia and can be detected in the plasma of nonpregnant and pregnant individuals. Levels of circulating EMAP-2 antigen are raised in pregnancy compared with nonpregnant controls; however, levels in patients with preeclampsia are identical to those in normal pregnant individuals. Conclusion: While circulating levels of EMAP-2 are increased in pregnancy, there is no evidence that EMAP-2 is involved directly in the pathogenesis of preeclampsia.